Old age is the single greatest risk factor for many diseases including heart disease, arthritis, cancer and dementia. By delaying the biological aging process, it may be possible to reduce the impact of age-related diseases, which could have great benefits for society and the quality of life of individuals. A drug called rapamycin, which is currently used to prevent organ rejection in transplant recipients, is a leading candidate for targeting aging. Rapamycin increases lifespan in several types of animals and delays the onset of many age-related conditions in mice.

Nearly all of the aging-related studies in mice have used the same dose of rapamycin given throughout the lives of the animals. Lifelong treatment with rapamycin wouldn’t be practical in humans and is likely to result in undesirable side effects. For example, the high doses of rapamycin used in transplant patients cause side effects including poor wound healing, elevated blood cholesterol levels, and mouth ulcers. Before rapamycin can be used to promote healthy aging in humans, researchers must better understand at what point in life the drug is most effective, and what dose to use to provide the biggest benefit while limiting the side effects.

Now, Bitto et al. show that treating mice with rapamycin for a short period during middle age increases the life expectancy of the mice by up to 60%. In the experiments, mice were given two different doses of rapamycin for only three months starting at 20 months old (equivalent to about 60-65 years old in humans). After receiving the lower dose, both male and female mice lived about 50% longer than untreated mice, and showed improvements in their muscle strength and motor coordination. When given the higher dose, male mice showed an even greater increase in life expectancy, but the female mice did not. These female mice had an increased risk of developing rare and aggressive forms of blood cancer, but were protected from other types of cancer.

Both drug treatments also caused substantial changes in the gut bacteria of the male and female mice, which could be related to effects of rapamycin on metabolism, immunity and health. More studies are needed to uncover precisely how such short-term treatments can yield long-term changes in the body, and how such changes are related to lifespan and healthy aging.

Successful interventions that increase healthy longevity in people could have profound benefits for quality of life, productivity, and reduced healthcare costs (Goldman et al., 2013; Kaeberlein et al., 2015). The drug rapamycin is a promising candidate for such an intervention, as it has been shown to increase lifespan in numerous species (Johnson et al., 2013) and to delay or reverse multiple age-associated phenotypes in mice including cognitive decline (Halloran et al., 2012; Majumder et al., 2012), cardiac dysfunction (Dai et al., 2014; Flynn et al., 2013), immune senescence (Chen et al., 2009), and cancer (Anisimov et al., 2011). Recently, a six week treatment with the rapamycin derivative RAD001 was reported to improve immune function in elderly people, as measured by response to influenza vaccine (Mannick et al., 2014), suggesting that at least some of the effects on aging in mice are conserved in humans. Despite these impressive results, the utility of rapamycin or other mTOR inhibitors to delay aging may be limited by side effects. The high doses of rapamycin and its derivatives used clinically to prevent organ transplant rejection are associated with adverse events, including impaired wound healing, edema, elevated circulating triglycerides, impaired glucose homeostasis, gastrointestinal discomfort, and mouth ulcers (Augustine et al., 2007; de Oliveira et al., 2011). While many of these side effects have not been observed in mice at the lower doses that extend lifespan, chronic treatment with encapsulated rapamycin (eRapa) in the diet at 14 ppm has been reported to cause gonadal degeneration in males, increased risk of cataracts, and impaired response to a glucose tolerance test (Wilkinson et al., 2012; Lamming et al., 2012).

Based on the premise that transient treatment with rapamycin during middle-age might be more suitable for clinical efforts to promote healthy aging than continuous treatment throughout life, we set out to investigate whether a single three-month treatment regimen can extend lifespan and healthspan in C57BL/6JNia mice starting at 20–21 months of age. We initially used a treatment regimen consisting of intraperitoneal (i.p.) injections of 8 mg/kg rapamycin daily for 90 days. This dose was selected because we have previously found that it increases survival and alleviates disease phenotypes in short-lived mouse models of dilated cardiomyopathy, muscular dystrophy, and the severe mitochondrial disease Leigh Syndrome (Ramos et al., 2012; Johnson et al., 2013). Based on efficacy in the Leigh Syndrome mouse model and serum drug levels in wild type mice, we estimate that this treatment regimen is comparable to dietary delivery of eRapa at approximately 378 ppm (Johnson et al., 2015), or 27-fold higher levels than initially shown to extend lifespan in mice when continuous treatment is initiated at either 9 months or 20 months of age (Harrison et al., 2009; Miller et al., 2011). Because prior studies have noted differences in the magnitude of lifespan extension following continuous rapamycin treatment in male versus female animals (Harrison et al., 2009; Miller et al., 2011, 2014), we examined the effect of this regimen in both sexes independently. Serum rapamycin levels did not differ significantly between male and female animals in our study (Figure 1—figure supplement 1).

During the three-month treatment period, we noted a significant decline in body weight of male mice receiving rapamycin injections relative to vehicle treated controls (Figure 1A), although food intake remained similar during the treatment (Figure 1—figure supplement 2). Decreased body weight persisted for several weeks following cessation of treatment (Figure 1A). This was accompanied by a striking increase in median life expectancy from the end of treatment of 60% (p=0.02, Figure 1B, Table 1) and an increase in overall median lifespan from birth of 16% (p=0.03, Figure 1—figure supplement 3, Table 2). This effect is larger than both the absolute and relative magnitude of lifespan extension resulting from continuous treatment to death with 14 ppm eRapa starting at around the same age in UMHET3 mice (Harrison et al., 2009). The longest-lived rapamycin-treated male in our cohort survived for 710 days post treatment to approximately 1400 days of age. Based on a survey of the literature, this is likely one of the longest-lived wild type C57BL/6 animals ever reported.

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This rapamycin regimen also resulted in a decline in body weight of female mice together with an increased trend of food intake in vehicle-treated animals with time (Figure 2A, Figure 2—figure supplement 2); however, we failed to detect a similar increase in post-treatment survival and lifespan in females (p=0.261, Figure 2B, Figure 2—figure supplement 1, Tables 1–2). Cox proportional hazards regression with robust standard errors and adjusting for cohort indicated significant evidence of a treatment difference between male and female mice (p=0.023). We speculated that this lack of lifespan extension results from an increase in aggressive hematopoietic cancers in the rapamycin treated females. Histopathological analysis showed that while 6/12 control females had round cell tumors (lymphoma and histiocytic sarcoma of hematopoietic origins), comparable to previous studies in C57BL/6 mice (Blackwell et al., 1995; Treuting et al., 2008), all the rapamycin-treated females examined (16 out of 16) had round cell tumors (Figure 3A,B and Figure 3—figure supplement 1, p=0.002). Additionally, an uncommon variant of lymphoma with plasmacytoid morphology affected 4 out of 16 mice examined in the rapamycin group and no vehicle treated mice (Figure 3A and Figure 3—figure supplement 1A,B). Round cell tumors affected more organs in rapamycin treated females, with multiple (≥2) organs affected in all of the rapamycin treated females compared to only 3 vehicle treated females (Figure 3C, p=0.01). Together, these data indicate a more aggressive phenotype of hematopoietic tumors in the high dose rapamycin treated females. In contrast, the incidence of non-hematopoietic neoplasms was dramatically decreased in the rapamycin group (Figure 3D). Nine non-hematopoietic neoplasms (5 pituitary adenomas, and 2 pulmonary adenomas, and 2 thyroid adenomas) were detected in 7 out of the 12 examined vehicle treated mice; whereas, only 1 out of 16 rapamycin treated females had non-hematopoietic neoplasia (1 pituitary adenoma) (Figure 3D, p=0.004).

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Both control and rapamycin treated male cohorts had a high incidence of systemic round cell neoplasia which affected ≥2 organs, and had an intravascular component and a morphology most consistent with histiocytic sarcoma, although immunohistochemistry was not performed (Figure 3—figure supplement 2). Several male mice also had eosinophilic (hyaline) protein droplets in the renal tubules consistent with lysozyme accumulation associated with histiocytic sarcoma (Hard and Snowden, 1991). Of the examined vehicle treated males, all (14 out of 14) had hematopoietic neoplasia affecting at least one organ. Additionally, 7 other types of neoplasia were detected on histopathology from the vehicle treated male group, including 1 gastric neoplasia, 1 thyroid adenoma, and 5 pulmonary adenomas. One male mouse had a subcutaneous facial mass characterized by sheets of round cells, most consistent with an extramedullary plasma cell tumor or mast cell tumor. Rapamycin treated males also had a high incidence of systemic round cell neoplasia affecting at least one organ (9 out of 13). In the rapamycin treated male group, 12 additional non-round cell tumors were detected on histopathology, including 1 hepatocellular carcinoma, 2 intestinal adenomas, 1 splenic hemangiosarcoma, 5 pulmonary adenomas, 1 pulmonary carcinoma, 1 thyroid adenoma, and 1 Zymbal’s gland adenoma (Figure 3—figure supplement 2). In the male group, no cases of systemic lymphoma with plasmacytoid morphology were seen. Although the number of mice examined is relatively small, based on these observations we speculate that this regimen of rapamycin treatment induced the detrimental side effect of aggressive hematopoietic cancers specifically in female mice, which occurred earlier in life and were sufficient to prevent lifespan extension in these animals despite other beneficial effects, such as reduced non-hematopoietic cancers. When data from both sexes are pooled together, daily injection of 8 mg/kg rapamycin for three months resulted in a non-significant (p=0.16) increase in life expectancy of 23% (Table 3). Between randomization and the end of treatment, 5 male mice (3 vehicle, 2 rapamycin) died of non-age related causes and were excluded from the analysis. Their inclusion in the analysis of survival does not significantly affect the results as described above (Figure 1—figure supplement 4).

In order to assess whether transient treatment with a lower dose and different delivery of rapamycin might reduce side effects in female mice and increase lifespan in both sexes, we utilized dietary eRapa at 126 ppm. Mice were fed a diet containing eRapa or the encapsulation control eudragit diet for 90 days starting at 20–21 months of age and then returned to a standard chow diet. In contrast with the effects of daily injection of 8 mg/kg rapamycin (Figures 1A and 2A), body weight and food intake were largely unaffected by this dietary rapamycin regimen (Figure 4 A,C,E, Figure 4—figure supplement 1), except for a transient, small increase in body weight in female mice fed eRapa, as determined by Student’s t test (Figure 4E). Median post-treatment life expectancy was significantly increased by 42% when female and male animals were considered together (p=0.002, Figure 4B, Table 3) and overall lifespan was increased by 13% (p=0.003, Figure 4—figure supplement 2A, Table 4). Similar results were observed stratifying on sex; three months of 126 ppm eRapa administration significantly increased post-treatment survival in both males and females independently (Figure 4D,F, Figure 4—figure supplement 2B,C and Tables 1–2). Cox proportional hazards regression with robust standard error estimates did not find evidence that sex modifies the treatment effect (p=0.904). Between randomization and the end of treatment, 1 male mouse died of non-age related causes and was excluded from the analysis. Its inclusion in the analysis does not significantly affect the results as described above (Figure 4—figure supplement 3A,B).

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In parallel with survival, we also examined healthspan parameters in these cohorts. The effects of rapamycin on age-associated decline in muscle function and motor coordination were assessed by testing forelimb grip strength and Rotarod performance. Assessments were performed in the same animals before onset of treatment, at the end of the 90 day treatment, and 90 days after the end of the treatment. While eudragit-fed mice showed a steady decline in both grip strength and Rotarod performance, rapamycin-fed mice scored significantly better than control mice in both assays after treatment (Figure 4G,H), suggesting that the healthspan promoting effect of rapamycin continues after treatment is discontinued. Furthermore, only rapamycin-fed mice showed a progressive increase in Rotarod performance during the 3 days of training and over the whole study, with mice performing better at 6 months compared to baseline (Figure 4H, large bracket).

In the course of routine animal husbandry, we noted that mice treated with either rapamycin regimen produced consistently smaller fecal pellets than age-matched controls (Figure 5A–D). This difference was present in both dry (Figure 5C,D) and freshly excreted feces (Figure 5—figure supplement 1A), indicating that water content is not a major factor affecting feces size. In addition, feces size was affected independently of the method of rapamycin delivery (Figure 5C,D), and the effect was persistent after cessation of treatment (Figure 5—figure supplement 1B,C). We hypothesized that changes in the microbiome may underlie this phenotype and therefore analyzed the fecal microbiome for each of the cohorts used in this study by deep-sequencing of bacterial 16S rRNA. Distance based permutation multivariate analysis of variance (MANOVA) (Anderson, 2001) indicated that rapamycin treatment induced a significant change in the composition of fecal microbiome (p=0.018 for injection cohorts. p=0.015 for feeding cohorts.p=0.005 for pooled cohorts), even after accounting for delivery method and batch effects (Figure 6—figure supplements 1–3).

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Among the most notable changes in fecal bacterial DNA content seen in the global microbiome analysis was a significant increase in prevalence of segmented filamentous bacteria (SFB, Candidatus Arthromitus sp.) in the rapamycin treated animals (Figure 6A, Figure 6—figure supplement 2A). SFB are intestinal Gram-positive bacteria with a segmented and filamentous morphology, and are not normally present at high levels in aged mice (Ericsson et al., 2014). The SFB genome lacks a majority of virulence factors and SFB are not invasive (Prakash et al., 2011); however, their tight adhesion to the intestinal epithelial cell induces differentiation of host immune cells (Atarashi et al., 2015). The increase in SFB following rapamycin treatment was confirmed by real-time PCR of DNA from fecal samples obtained from both mice receiving injections or encapsulated rapamycin (Figure 6B, Figure 6—figure supplement 2B), as well as by semi-quantitative histological scoring of the small intestine in an independent cohort of mice obtained from the Harrison Lab at the Jackson Laboratory and injected with 8 mg/kg/day of for 3 months at the University of Washington (Figure 6C,D, Figure 6—figure supplement 2C). Since increased SFB DNA was observed both in mice injected with rapamycin and mice fed dietary eRapa, this effect is independent of mode of drug delivery. To the best of our knowledge, this represents the first pharmacological intervention to increase SFB in any animal. It will be of interest to determine whether these and other effects of rapamycin on the microbiome are shared across species and play any causal role in the beneficial or detrimental effects of this drug.

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Taken together, our data demonstrate that a single three-month regimen of rapamycin is sufficient to robustly increase life expectancy in middle-aged mice, comparable to the effects previously reported for life-long treatment, while also improving measures of healthspan and substantially altering the microbiome. This work extends prior evidence indicating that short-term rapamycin treatment can improve health in mice, including one experiment suggesting that 4 mg/kg rapamycin every other day for 6 weeks enhanced survival until around 30 months of age in a small cohort (Chen et al., 2009), and studies reporting improvements in cardiac (Dai et al., 2014; Flynn et al., 2013) and immune (Chen et al., 2009) function following transient treatment with rapamycin. In the animals treated with the 126 ppm eRapa diet in this study, the improvements in lifespan and health were achieved without overt detrimental side effects, although it is possible that some side effects were undetected, and we did not explicitly test for cataracts, gonadal degeneration, and other adverse outcomes. In the case of the 8 mg/kg/day injection regimen, serious side effects were noted in female, but not male mice. Intriguingly, a dramatic shift toward aggressive hematopoietic cancers and away from non-hematopoietic cancers was observed in these female mice. This is consistent with a similar weak trend seen in kidney transplant patients receiving rapamycin to prevent organ rejection, suggesting a possible conservation of mechanism and clinical relevance (Mathew et al., 2004). Our data indicate a need to carefully consider sex effects when optimizing treatment regimens and mechanism of drug action. They also illustrate the importance of better understanding the effects of mTOR inhibitors on differential cancer risk, particularly as mTOR inhibitors are being tested and used clinically for a variety of purposes including the treatment of some rare forms of cancer. The importance of evaluating potential risks and adverse side effects when developing interventions to promote healthy aging should not be underestimated.

This study extends other recent work aimed at developing mid-life interventions to promote healthy aging. Of particular note are two studies reporting improved healthspan from short-term treatments in older mice with a JAK pathway inhibitor (Xu et al., 2015) and increased lifespan after transient treatment with the NAD⁺ precursor nicotinamide riboside (Zhang et al., 2016). From a translational perspective, a healthy aging intervention that can be applied for a relatively short period of time during mid- or late-life is likely to have advantages in cost, practicability and quality of life of people, and we look forward to further developments in this area.

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Author details

1. Alessandro Bitto

   Department of Pathology, University of Washington, Seattle, United States

   Contribution

   AB, Designed experiments, Wrote the paper, Performed drug injections for mice for semi-quantitative grading of SFB, Performed lifespan experiments on feeding models, Performed rotarod assay and forelimb grip strength test

   Contributed equally with

   Takashi K Ito

   Competing interests

   The authors declare that no competing interests exist.

2. Takashi K Ito

   Department of Pathology, University of Washington, Seattle, United States

   Contribution

   TKI, Conceived the project, Designed experiments, Wrote the paper, Performed lifespan experiments on injection models, Performed analysis on fecal sample size, Performed microbiome analysis, Performed the quantitative analysis of SFB with real-time PCR

   Contributed equally with

   Alessandro Bitto

   Competing interests

   The authors declare that no competing interests exist.

3. Victor V Pineda

   Department of Pathology, University of Washington, Seattle, United States

   Contribution

   VVP, Performed the quantitative analysis of SFB with real-time PCR, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

4. Nicolas J LeTexier

   Department of Pathology, University of Washington, Seattle, United States

   Contribution

   NJL, Performed analysis on fecal sample size, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

5. Heather Z Huang

   Department of Pathology, University of Washington, Seattle, United States

   Contribution

   HZH, Performed lifespan experiments on injection models, Drafting or revising the article, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

6. Elissa Sutlief

   Department of Pathology, University of Washington, Seattle, United States

   Contribution

   ES, Performed drug injections for mice for semi-quantitative grading of SFB, Performed lifespan experiments on feeding models, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

7. Herman Tung

   Department of Pathology, University of Washington, Seattle, United States

   Contribution

   HT, Performed drug injections for mice for semi-quantitative grading of SFB, Performed lifespan experiments on feeding models, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

8. Nicholas Vizzini

   Department of Pathology, University of Washington, Seattle, United States

   Contribution

   NV, Performed drug injections for mice for semi-quantitative grading of SFB, Performed lifespan experiments on feeding models, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

9. Belle Chen

   Department of Pathology, University of Washington, Seattle, United States

   Contribution

   BC, Performed lifespan experiments on feeding models, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

10. Kaleb Smith

    Department of Pathology, University of Washington, Seattle, United States

    Contribution

    KS, Performed lifespan experiments on feeding models, Drafting or revising the article

    Competing interests

    The authors declare that no competing interests exist.

11. Daniel Meza

    Department of Pathology, University of Washington, Seattle, United States

    Contribution

    DM, Performed lifespan experiments on feeding models, Drafting or revising the article

    Competing interests

    The authors declare that no competing interests exist.

12. Masanao Yajima

    Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, United States

    Contribution

    MY, Performed microbiome analysis, Performed statistical analysis and designed the figures on microbiome analysis, Drafting or revising the article, Contributed unpublished essential data or reagents

    Competing interests

    The authors declare that no competing interests exist.

13. Richard P Beyer

    Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, United States

    Contribution

    RPB, Performed statistical analysis and designed the figure on rotarod assay and part of lifespan analysis, Drafting or revising the article, Contributed unpublished essential data or reagents

    Competing interests

    The authors declare that no competing interests exist.

14. Kathleen F Kerr

    Department of Biostatistics, University of Washington, Seattle, United States

    Contribution

    KFK, Performed statistical analysis and designed the figure on rotarod assay and part of lifespan analysis, Drafting or revising the article, Contributed unpublished essential data or reagents

    Competing interests

    The authors declare that no competing interests exist.

15. Daniel J Davis

    Department of Veterinary Pathobiology, University of Missouri, Columbia, United States

    Contribution

    DJD, Performed microbiome analysis, Performed the quantitative analysis of SFB with real-time PCR, Drafting or revising the article

    Competing interests

    The authors declare that no competing interests exist.

16. Catherine H Gillespie

    Department of Veterinary Pathobiology, University of Missouri, Columbia, United States

    Contribution

    CHG, Performed microbiome analysis, Provided intellectual input in SFB analysis, Drafting or revising the article

    Competing interests

    The authors declare that no competing interests exist.

17. Jessica M Snyder

    Department of Comparative Medicine, University of Washington, Seattle, United States

    Contribution

    JMS, Wrote the paper, Performed histopathological analysis and semi-quantitative grading of SFB

    Competing interests

    The authors declare that no competing interests exist.

18. Piper M Treuting

    Department of Comparative Medicine, University of Washington, Seattle, United States

    Contribution

    PMT, Designed experiments, Performed histopathological analysis and semi-quantitative grading of SFB, Provided intellectual input in SFB analysis, Drafting or revising the article

    Competing interests

    The authors declare that no competing interests exist.

19. Matt Kaeberlein

    Department of Pathology, University of Washington, Seattle, United States

    Contribution

    MK, Conceived the project, Designed experiments, Wrote the paper, Managed and supervised the whole project

    For correspondence

    kaeber@uw.edu

    Competing interests

    The authors declare that no competing interests exist.

    "This ORCID iD identifies the author of this article:" 0000-0002-1311-3421

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